This invention is an improvement in a known chemical process. More specifically, it concerns an improved process for making polypeptides by the coupling reaction of an amino acid azide with an amino acid compound in a substantially anhydrous reaction system.
The azides of carboxylic acids are useful chemical intermediates in processes for converting the acids into other types of compounds. For example, the Curtius rearrangement transforms an azide into the corresponding isocyanate for use in making urethanes, ureas, amides or amines. One synthesis of .alpha.-amino acids proceeds through the azide of an .alpha.-cyano acid as an intermediate. Azides are of particular interest in the Bergmann synthesis of polypeptides by the coupling reaction of an N-protected amino acid azide with a C-protected amino acid such as the ester, amide, N-substituted amide, or a solvent-soluble salt.
The latter synthesis, in particular, is of interest because of the increasingly intensive work in the field of synthetic polypeptides. These compounds offer a means for understanding the structure and mode of participation of more complex natural polypeptides and proteins in life processes and "tailor-made" polypeptides differing from naturally occurring molecules in predetermined ways provide useful new tools in biochemical and medical research. Complex, naturally occurring polypeptides such as secretin, insulin, and calcitonin have recently been synthesized in the laboratory using a progression of coupling reactions.
The Bergmann synthesis couples amino acid molecules by reacting the amino group of a carboxyl-protected amino acid compound such as the ester, amide, or salt with the acyl azide of an amino acid or peptide wherein the amino group or groups of the azide reactant are protected against undesirable side reactions by an easily removable N-protective group such as an acyl group, a toluenesulfonyl group or a urethane radical.
Generally in azide-amino acid couplings, the azide is made from the corresponding hydrazide by treatment with nitrous acid in aqueous acid (e.g., sodium nitrite plus dilute hydrochloric acid) or with an alkyl nitrite in an acidic organic medium. In both cases the azide solution must be washed with base (e.g., bicarbonate solution) to remove excess acid. This step may tend to cause racemization at the .alpha.-carbon atom of the amino acid or peptide azide. After washing with base, the azide solution is dried prior to reaction with the amino acid compound. Hence, during these manipulations which are time consuming and which must be conducted in the cold, there is greater possibility of rearrangement of the azide to the corresponding isocyanate, a side reaction which may lead to the formation of an urea instead of peptide by the following sequence of reactions: ##STR1## There are special examples in the literature of azide preparations in which, (a) a nonpolar solvent is used in conjunction with an alkyl nitrite as the nitrous acid source with the same reaction vessel and used for both the nitrosylation and coupling reactions excess acid is neutralized with an organic tertiary base; or (b) a polar solvent such as dimethylformamide is used with an inorganic nitrite and hydrochloric acid, to supply nitrous acid, a single reaction vessel is used, and excess acid is neutralized by an organic tertiary base. The present invention, however, has the advantage over the former of using an inexpensive nitrosylating source and over the latter of allowing the use of a nonpolar solvent.
So-called crown complexes of inorganic compounds with macrocyclic polyethers are described by Pedersen in U.S. Pat. No. 3,562,295. These complexes are generally suggested for use in extraction processes and diazotization and nitrosylation reactions although no examples of the latter are given and there is no mention of their possible use in anhydrous polypeptide preparations.